Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease, because it disrupts normal protective gut flora and enables C. difficile to colonize the colon. C. difficile damage host tissu by secreting toxins and disseminates by forming spores. The toxins encoding genes, tcdA and tcdB are part of a pathogenicity locus, which also carry the gene tcdR that codes for the toxin genes positive regulator. TcdR is an alternate sigma factor that initiates transcription at tcdA an tcdB promoters. Alternate sigma factors are known to regulate virulence and virulence associated genes in many pathogenic bacteria. Mutation in tcdR affected both toxin production and sporulation in C. difficile. Transcriptome analyses revealed many differentially expressed sporulation genes in tcdR mutant. First aim of this project will test the role of TcdR in C. difficle sporulation and the second aim will characterize mutations in tcdR upstream that are important for its transcription. Aims of this study promises to provide critical information on gene regulatoy networks that are important for C. difficile pathogenesis. These findings will positively impact th pursuit of novel therapeutics to treat C. difficile infections.